This paper provides a new method to compare and then reveal the vacancy sink efficiencies quantitively between different hetero-interfaces with a shared Cu layer in one sample, in contrast to previous studies, which have compared the vacancy sink efficiencies of interfaces in different samples. Cu-Nb-Cu-V nanoscale metallic multilayer composites (NMMCs) containing Cu/V and Cu/Nb interfaces periodically were prepared as research samples and bombarded with helium ions to create vacancies which were filled by helium bubbles. A special Cu layer shared by adjoining Cu/V and Cu/Nb interfaces exists, in which the implanted helium concentration reaches its maximum and remains nearly constant with a well-designed incident energy. The results show that bubble-denuded zones (BDZ) close to interfaces exist, and that the width of the BDZ close to the Cu/V interface is less than that of Cu/Nb interface. This result is explained by one-dimensional diffusion theory, and the ratio of vacancy sink efficiency between Cu/V and Cu/Nb interfaces is calculated. Conclusively, Cu/Nb interfaces are more efficient than Cu/V interfaces in eliminating vacancies induced by radiation. Nanoscale metallic multilayer composites (NMMCs) have been widely studied due to their ultra-high strengths and enhanced radiation damage tolerance. During the last decade, a number of NMMCs were fabricated and researched, including Cu/Nb ,2,3, Cu/Cu/Nb and Cu/V NMMCs have two things in common. The first is that the Kurdjumov-Sachs (K-S) orientation relationship  exists i−5 Pa was reached prior to deposition and argon partial pressure during sputtering was kept at 0.5 Pa. The deposition rate was approximately 0.2 nm/s.Cu-Nb-Cu-V NMMCs were prepared using the DC magnetron sputtering technique at room temperature on Si (100) substrates with individual layer thickness of 48 nm and total thickness of 576 nm (referred to as Cu-Nb-Cu-V 48 nm thereafter). In the deposition process, a base pressure of 4 × 1016 ions/cm2. Reasonably, the target model in SRIM simulation for Cu-Nb-Cu-V 48 nm NMMCs is the multilayered model, as The Stopping and Range of Ions in Matter (SRIM)  computer16 ions/cm2 was conducted at room temperature to make sure that the He concentration exceeded a critical value so that the defect clusters could be observed in TEM, as described in previous studies [Helium ions were chosen to radiate samples to create point defects inside with an incident energy of 119 keV. The injection dose of 6 × 10The depth distributions of helium concentration (DHC) and displacements per atom (dpa) in Cu-Nb-Cu-V 48 nm NMMCs are predicted by SRIM, as The in-focused XTEM micrographs of as-deposited Cu-Nb-Cu-V 48 nm NMMC are presented in 16 ions/cm2. Bubbles are observed in the region of 7th to 11th layers. These bubbles filled by helium atoms may be imaged by structure factor contrast under dynamical or bright-field kinematical imaging conditions [nditions . The con16 ions/cm2. The existence of the bubbles is confirmed and indicated by arrows. As we can see, bubbles are observed in Layer 9-Cu, but not in the 8-V and 9-Nb layers. Meanwhile, two zones free of bubbles in Layer 9-Cu close to the Cu/Nb and Cu/V interfaces exist. These zones, depleted of defect clusters near the interfaces, are referred to as ”bubble denuded zones” (BDZs) [” (BDZs) ,21. The In order to compare the sink efficiencies of Cu/V and Cu/Nb interfaces in the same sample for the first time, Cu-Nb-Cu-V NMMCs containing Cu/V and Cu/Nb interfaces are composed in a single sample for this paper. Previous studies have stated that the interfaces play a big role in the distribution of defect clusters in their vicinity zones ,21. TherThe most primary form of atomic damage sustained by an irradiated material is point defects, including vacancies and interstitials. These interstitials within the intermediate Cu layer after irradiation include helium atoms and disordered Cu atoms. As helium ion irradiation occurs over time, point defects in NMMCs gradually evolve into defect clusters. For Cu-Nb-Cu-V 48 nm NMMCs, bubbles are observed at room temperature, as eqc is the vacancy concentration under thermal equilibrium conditions and In the formation stage of helium bubbles, vacancies are firstly formed and then combined with helium atoms into a stable cluster. These clusters may build up by capturing vacancies and helium atoms, and gradually become detectable large bubbles. Hence, the difference in width of these two BDZs originates from different vacancy sink efficiencies of interfaces. Following a previous study , the vacns model  with theBecause the BDZ close to Cu/Nb interface and the BDZ close to Cu/V interface exist in the same Cu layer, the vacancy concentration in the BDZ/Cu interface near Cu/Nb interface takes the same value of Note that the numerators of these two sink efficiencies are exactly same. So, the ratio of sink efficiencies of these two interfaces is:The rate of recombination ×106 s−1 . The vac9 m2 s−1 . Taking Conclusively, the Cu/Nb interface had a higher sink efficiency than that of the Cu/V interface. The difference in vacancy sink efficiency for Cu/V and Cu/Nb interfaces may be attributed to defect trapping sites in them. Misfit dislocation interactions (MDIs) have been shown to serve as preferential vacancy trapping sites at some hetero-interfaces in previous studies. And the Cu/Nb interface contains a 5 times greater density of MDIs than Cu/V . TherefoIn this paper, a new method to quantitively compare the vacancy sink efficiencies between different interfaces is proposed. This method is the first to integrate the comparison in the same sample, in contrast to the previous studies which have compared two interfaces existing in two individual samples. The average width of bubble-denuded zones was measured to be 2.5 ± 0.2 nm for the Cu/V interface and 3.5 ± 0.3 nm for the Cu/Nb in our designed experiment. Based on one-dimensional diffusion theory, the ratio of vacancy sink efficiencies of Cu/V and Cu/Nb interfaces was shown to be approximately 95% of Cu/Nb, indicating that Cu/Nb interfaces are slightly more efficient than Cu/V interfaces in eliminating point defects and associated He bubbles induced by radiation.


Metastatic colorectal cancer (mCRC) is a highly heterogeneous disease from a clinical, molecular, and immunological perspective. Current predictive models rely primarily in tissue based genetic analysis, which not always correlate with inflammatory response. Here we evaluated the role of a circulating inflammatory signature as a prognostic marker in mCRC.Two hundred eleven newly diagnosed patients with mCRC were enrolled in the study. One hundred twenty-one patients had unresectable metastases, whereas ninety patients had potentially resectable liver metastases at presentation. Analysis of miR-21, IL-6, and IL-8 in the plasma of peripheral blood was performed at baseline. Patients with high circulating levels of ≥2 of the three inflammation markers  were considered to have the “Inflammation phenotype-positive CISIG”.p = 0.001 HR 2.09, 95% CI 1.2–3.67) for positive vs. negative CISIG. In contrast, the individual components were not significant. In the same population the median overall survival was 46.2 compared to 66.0 months  for positive vs. negative CISIG, but not significant for the individual components. In the unresectable population, the median overall survival was 13.5 compared to 25.0 months  for positive vs. negative CISIG. IL-6 was independently prognostic with overall survival of 16.2 compared to 27.0 months  for high vs. low IL-6, but not the other components. Using a Cox regression model, we demonstrated that CISIG is an independent predictive marker of survival in patients with unresectable disease .Positive CISIG was found in 39/90 (43%) and 50/121 (45%) patients in the resectable and unresectable cohort, respectively. In the resectable population the median relapse-free survival was 18.4 compared to 31.4 months (In two different cohorts, we demonstrated that CISIG is a strong prognostic factor of relapse-free and overall survival of patients with mCRC. Based on these data, analysis of circulating inflammatory signaling can be complimentary to traditional molecular testing. In contrary to studies performed on tissue samples, serum levels of miR-21 alone have limited prognostic value.15The association of chronic inflammation and CRC tumorigenesis is well established and in the last decade has received growing supportive evidence from epidemiological, genetic, and pharmacological studies. The crosstalk between inflammatory, stromal, and cancer cells occurs in multiple levels allowing the tumor cells to undergo genetic, epigenetic, and biochemical changes to acquire an aggressive phenotype. MicroRNAs are a critical component of this cycle. miR-21 is specifically relevant to CRC tumorigenesis.Tissue-based analyses have not always well reflected the current state of the tumor-host interactions. Indeed, cancer cachexia, systemic inflammatory response, and tumor-associated symptom burden are poorly evaluated by studies of the tumor tissue. Additionally, tumor-based assessments are limited to a single time-point in the disease course and do not fully reflect the current state of the disease. Circulating markers, in contrast, provide temporal relevance and integrate a systemic response to the tumor.On the basis of this rationale, in this study, we examined the role of Circulating Inflammation SIGnature (CISIG) determined by high miR-21, IL-6, and IL-8 circulating levels as a potential marker in metastatic CRC. In two different cohorts, we demonstrated that CISIG is a strong prognostic factor of progression-free and overall survival of patients with metastatic CRC. Therefore, the inflammatory signature might be valuable tool in secondary prevention and patient selection for aggressive medical management.Two hundred eleven 211) newly diagnosed patients with metastatic colorectal cancer who presented to MD Anderson Cancer Center from April 2002 to December 2008 were enrolled in the study. The patients were divided into two cohorts based on clinical characteristics. The first cohort included one hundred twenty-one (121) patients with unresectable metastases at presentation, whereas the second validation cohort included ninety patients (90) with potentially resectable metastases at presentation. Patients from the second cohort subsequently underwent surgical intervention including partial hepatectomy, with curative intent within 2 years of the date of plasma draw. Patient characteristics are shown in Table 1 newly dHuman plasma samples for CISIG analysis were obtained after informed consent had been obtained from the patient, per Institutional Review Board (IRB) protocol at the MD Anderson Cancer Center (MDACC). Samples were drawn at the time of presentation, and before initiation of any systematic therapy.Progression-free survival (PFS) was defined as the interval between enrollment on the study and clinical, laboratory or radiologic progression, or death from any cause. Relapse-free survival (RFS) was defined as the interval between liver resection and recurrence in liver or elsewhere, or death from any cause. Overall survival (OS) was defined as the interval between enrollment on the study and death from any cause.Plasma samples were drawn and collected in ethylenediaminetetraacetic acid tubes and centrifuged at 2800 r.p.m. for 10 min at −22 °C. Sample aliquots were placed into 0.5-mL cryovials and stored at −70 °C to −80 °C until analysis. Plasma samples  were thawed in parallel and each used for suspension bead multiplex assays.−ΔΔCt method with 95% confidence intervals. Circulating miR-21 levels were determined using levels of mir-16 as a control . All hemolytic samples  were excluded from the study.For microRNA-based RT-PCR assays, 2.5 µL of enriched small RNAs from plasma samples were reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit  according to manufacturer’s instructions in a total reaction volume of 7.5 µL. A 1∶20 dilution of RT products was used as template for the PCR stage. PCR reaction was performed in triplicate wells using TaqMan 2× Universal PCR Master Mix with conditions as described previously. No-template controls for both RT step and PCR step were included to ensure target specific amplification. The 7900 Sequence Detection System 2.3 (Applied Biosystems) software defaults were used to compute the relative change in RNA expression by the 2Levels of cytokines and biomarkers in serum were assessed using multiplex bead assay . Briefly, samples were incubated for 1 h on the array plates that were pre-spotted with capture antibodies specific for each protein biomarker. Plates were decanted and washed four times before adding a cocktail of biotinylated detection antibodies to each well. After incubating with detection antibodies, plates were washed four times and incubated with streptavidin-horseradish peroxidase conjugate. All incubations were done for 30 min at room temperature with shaking at 200 rpm. Plates were again washed before adding a chemiluminescent substrate, followed by immediate imaging. Cytokine array data are available upon request to the corresponding author.3 For IL-6 and IL-8, the median was used as a cut-off point to dichotomize the levels of these markers to high vs. low. Patients with high circulating levels of >2 of the 3 inflammation markers  were considered to have the “Inflammation phenotype-positive CISIG”. Spearman correlation coefficients for correlation between circulating miR-21, IL-6, and IL-8 were computed. Median OS, and disease-free survival were estimated non-parametrically using the Kaplan–Meier method and compared by the log-rank test. Cox proportional hazards regression was used to adjust for potential confounders and significant differences were assessed using the log-rank test. Calculations were performed with SPSS-version 23.0 software  and SAS 9.4 (SAS Institute). p values of less than 0.05 were considered statistically significant. The OS and corresponding censoring were computed in months from diagnosis to death for each patient. Additionally, progression-free survival and recurrent-free survival was computed for patients and was defined as the interval between blood draw and recurrent disease.Statistical analysis was performed using STATA version 11.0. The cut-off of the highest tertile was used to define high level of circulating miR-21 and the bottom two tertiles were defined as low level, based on previous literature.p > 0.05)  was found in 39/90 (43%) and 50/121 (45%) patients in the resectable and unresectable cohort, respectively. The difference in CISIG prevalence between the two cohorts was not statistically significant (p < 0.001). In the integrated study population (211 patients), the Spearman’s correlation coefficient between miR-21/ IL-6, miR-21/ IL-8, and IL-6/ IL-8 was 0.38, 0.38, and 0.76 respectively (data not shown).The correlation between inflammatory markers was performed using Spearman’s coefficient. In agreement with previous studies performed in CRC tissue samples, the serum levels of miR-21, IL-6, and IL-8 showed statistically significant correlation  for positive vs. negative CISIG. In contrast, the individual components were not significant: 19.7 compared to 24.2 months  for high vs. low miR-21, 18.4 compared to 28.7 months  for high vs. low IL-6, and 21.7 compared to 24.2 months  for high vs. low IL-8, and  for positive vs. negative CISIG, but not significant for the individual components: 46.2 compared to 66.0 months  for high vs. low miR-21, 46.2 compared to 66.4 months  for high vs. low IL-6, and 46.2 compared to 62.9 months  for high vs. low IL-8  for positive vs. negative CISIG. IL-6 was independently prognostic with overall survival of 16.2 compared to 27.0 months  for high vs. low IL-6, but not the other components: 19.7 compared to 24.2 months  for high vs. low miR-21, and 19.1 compared to 24.3 months  for high vs. low IL-8 , Hgb < 10 g/dL, CEA, NLR > 5, previous cytotoxic chemotherapy, albumin < 3.5 g/dL, platelets > 310 000 (cells/mcL), and WBC > 8000 (cells/mcL) as covariates. After adjusting for the above covariates, the inflammatory signature was still significantly predictive of overall survival . The only other significant predictor in the model was left sided tumor  , elevated WBC, elevated platelets, and previous chemotherapy are associated with poor prognosis in metastatic colorectal cancer.Individual inflammatory markers and cytokines have previously shown prognostic value in solid tumors in small trials. In this study, we demonstrate that a signature of circulating inflammatory markers correlates with metastatic colorectal cancer aggressiveness. Of note, the prognostic value of the CISIG is evaluated in both patients with resectable and unresectable tumors with similar statistically significant results.18 In parallel, the prognostic impact of BRAFV600E mutations in metastatic CRC is also dramatic, since patients with BRAFV600E-mutant CRC survive approximately less than half as long as patients with BRAF wild-type (WTBRAF) metastatic disease.19 In contrary, BRAF mutations that occur outside the codon 600 define a clinically distinct phenotype of metastatic CRC with excellent prognosis.20 In a recent study, we demonstrated that the double mutation of APC and PIK3CA in tissue samples from liver metastases was associated with reduced RFS and OS rates, independently of Ras mutation status and surgical management.21 However, these features do not correlate well with the inflammatory response of the host to the tumor. As such, analysis of inflammatory signaling can be complimentary to traditional molecular testing.A number of studies demonstrated a correlation between genetic mutations and aggressive disease. The association of Ras mutations with poor disease outcomes after hepatectomy is well documented.27 In parallel, activation of NF-kB/IL-6 paracrine axis promotes proliferation of CRC epithelial cells,29 whereas the IL-6-STAT3-mir-34a signaling pathway enhances the EMT phenotype.30 In agreement with the above findings, the Src/NF-kB/IL6 axis has been implicated in epithelial transformation in a positive feedback loop.31 Similarly, the IL-8-CXCR2 autocrine/paracrine axis has been implicated in proliferation of epithelial cells, EMT-phenotype, and VEGF-independent angiogenesis.32 Furthermore, it was proved, in a lung cancer model, that miR-21 can function as ligand to human Toll-like receptor (TLR) family, murine TLR8 in immune cells, triggering a TLR-mediated pro-metastatic inflammatory response including the secretion of cytokines such as TNF-α and IL-6, that ultimately may lead to tumor growth and metastasis.33The mechanism of miR-21, IL-6, and IL-8-induced CRC progression is not clearly understood. Studies performed in preclinical models showed that miR-21 downregulates tumor-suppressor genes including PTEN, and causes upregulation oncologic pathways such us RhoB.34 Based on these results, an inflammatory signature might be valuable in distinguishing CMSs and tailor therapy in patients with metastatic CRC.Poor outcomes and driving processes including EMT, stemness, and angiogenesis have been recapitulated in comprehensive gene expression studies such as the consensus molecular subtypes (CMS). The association of distinct CMSs with tumor inflammation and systemic inflammatory response is currently under evaluation. An initial report demonstrated that out of the four CMSs, two highly express immune-specific gene signatures. The good-prognosis microsatellite unstable (CMS1) is characterized by overexpression of genes specific to cytotoxic lymphocytes. In contrast, the poor-prognosis mesenchymal subgroup (CMS4) expresses markers of lymphocytes and of cells of monocytic origin.35 Similarly, an alternate analysis comparing the most differentially expressed miRNAs to each CRC subtype demonstrated that these miRNAs also defined the mesenchymal phenotype.36 Both studies also found that downregulation of miR-200 (and family members) contributed significantly to regulating EMT, matrix remodeling, and TNF signaling via NF-kB. A master regulator function for miRNA is suggested by these findings and supports inclusion of miRNAs in future prognostic scores.MicroRNA may have a role in explaining groups defined by comprehensive gene expression in CRC. Network approaches combining transcription factor, methylation, and miRNA analysis demonstrate that up to 74.8% of variation seen in gene expression in mesenchymal vs. other subtypes is explained by miRNAs.While this study demonstrates the potential applications of circulating inflammatory markers to determine aggressiveness of metastatic tumors and responses to treatment, it must be interpreted within the contexts of its limitations. Due to the methodology used, it lacks the external validity of cytokine assays owing to use of percentile cut-offs. Also, this study lacks the direct correlation of circulating markers with expression of the same markers in tissue samples from matched cases. Our assessment of inflammatory markers and clinical characteristics was blinded and samples were prospectively collected, both features that add to the validity of the experimental design but do not obviate the need for direct association of circulating markers with tissue marker expression.In summary, we demonstrated that a three-marker circulating inflammation signature could be feasible and predictive of outcomes with standard systemic therapy and overall survival, and more importantly demonstrate the potential as a noninvasive assessment of the inflammatory state of the tumor.additional informationSupplementary table 2Supplementary Table 1Figure legends


Analysis of the drug release mechanism revealed the diffusion-controlled release of LAP and anomalous transport of PTX. Drug content analysis in lyophilized micelles and micellar solution showed their good storage stability for at least 6 weeks. Blank micelles, LAP-loaded micelles and free LAP did not affect MCF-7 breast cancer cell proliferation, suggesting that the cytotoxicity of PTX-, PTX/LAP-loaded micelles, and the binary mixture of free PTX and LAP was solely caused by PTX. PTX/LAP-loaded micelles showed greater toxicity compared to the binary mixture of PTX and LAP after 48 h and 72 h. Only free PTX alone induced P-gp activity. This study showed the feasibility of using a LAP and PTX combination to overcome MDR in MCF-7 cells, particularly when co-loaded into micelles. We suggest that PTX/LAP micelles can be applicable not only for the therapy of HER-2-positive, but also HER-2-negative breast cancers.To find better strategies to enhance the cytotoxic effect of paclitaxel (PTX) and lapatinib (LAP) against breast cancer cells, we analyzed the efficacy of a novel delivery system containing polylactide-co-poly(ethylene glycol) (PLA-PEG) filomicelles of over 100 nm in length and spherical micelles of approximately 20 nm in diameter. The  Despite significant progress in the last few decades in breast cancer treatment and the use of different therapeutic strategies, this cancer remains a serious clinical problem due to the nature of its proliferation and loss of sensitivity to pharmacological agents .Paclitaxel (PTX) is used to treat breast cancer both as a monotherapy and in combination with other anticancer drugs depending on the severity of the cancer, the presence of metastases and preceding therapeutic management. It is characterized by high effectiveness both in early breast cancer and in metastatic breast cancer . The priIn recent years, there are an increasing number of papers dealing with a lack of efficacy or the decreased efficacy of various drugs used in anticancer treatment. One of the frequently suggested strategies of overcoming this serious problem is the development of new drug dosage formulations which contain single drugs or their combinations. These novel drug delivery systems include polymeric nanoparticles as well as liposomes. The antitumor efficacy of such systems shows a cellular toxicity similar to that of the free compounds or can be markedly increased. PTX is one of the drugs that are most often incorporated into nanoparticles. Cosco et al. , using aIn this study, we analyzed the cytotoxic effect of PTX and LAP on MCF-7 breast cancer cells, which are characterized by the absence of EGFR and HER-2 receptors. To find better strategies to overcome MDR, we analyzed the efficacy of PTX- and LAP-loaded micelles. It seems that such a micellar delivery system, which combines different strategies to overcome the efflux-mediated MDR, would be more effective for the treatment of resistant breast cancer than free PTX and LAP. Polylactide-co-poly(ethylene glycol) (PLA-PEG) was used to prepare micelles for the co-delivery of PTX and LAP. PEG is the most commonly used to form the hydrophilic shell of polymeric micelles, because it is biocompatible, reduces micelle aggregation and prevents interaction with serum proteins ,23. PLA n of both the polylactide (PLA) and poly(ethylene glycol) (PEG) blocks was 5000. PTX was purchased from LC Laboratories . LAP was purchased from Sigma-Aldrich .Polylactide-co-poly(ethylene glycol) (PLA-PEG) diblock copolymer was synthesized according to the procedure described before . The Mn g for 5 min  to eliminate the unloaded drug. The supernatant was recovered, lyophilized and stored at 4 °C for further analysis. PLA-PEG micelles loaded with paclitaxel and lapatinib (PTX/LAP) were prepared by the co-solvent evaporation method. The ratio of drugs to polymer was 10% (w/w) and each drug was used in equal amounts. PLA-PEG with LAP was dissolved in methylene chloride, followed by addition of distilled water to obtain a concentration of 1 mg/mL. The mixture was stirred vigorously at room temperature for 3 h and left for solvent evaporation for 24 h. Then, PTX dissolved in ethanol was added to the micelle solution and the mixture was stirred for 3 h, followed by centrifugation at 12,300× ®VWR/Hitachi, Tokyo, Japan). Measurements were carried out with the use of a LiChroCART® 150 × 4.6 mm Purospher® STAR RP-18e (5 μm) column  at a temperature of 25 °C at 227 nm. The mobile phase consisted of acetonitrile and water (70:30) at a flow rate of 0.5 mL/min.For the determination of the loading content (LC) and encapsulation efficiency (EE), lyophilized micelles were dissolved in acetonitrile and analyzed by high-performance liquid chromatography  spectroscopy was performed on a Bruker-Avance II Ultrashiels Plus spectrometer (600MHz) using DMSO6-d as a solvent. Chemical shifts (δ) were given in ppm using tetramethylsilane as an internal reference. Proton nuclear magnetic resonance . Images were recorded on the Eagle 4k HS camera  and processed with TIA software . The micellar solution was filtered through a 0.85-μm filter and placed on a copper grid covered with carbon film and stained negatively (with the use of 2% phosphotungstic acid (PTA)) and air dried at room temperature before measurements. 2 (adjusted coefficient). The in vitro drug release was analyzed by the dialysis method with the use of 3-mL Slide-A-Lyzer dialysis cassette  with a MWCO of 3500 Da. Lyophilized drug-loaded micelles were dispersed (1 mg/mL) in pH 7.4 phosphate buffered saline (PBS) and added into a dialysis cassette. Each cassette was placed in 400 mL PBS (pH 7.4). A sample of 50 μL was drawn from each cassette at 1 h, 24 h, 48 h, etc. and replaced by the same volume of fresh PBS. At the same time, the PBS was changed to ensure sink conditions. The amount of the released drugs was measured by HPLC. DDsolver, an Excel Ad in Program , was useg for 5 min and the supernatant was lyophilized, dissolved in acetonitrile and analyzed by means of HPLC. Lyophilized micelles were reconstituted in acetonitrile, filtered through a 0.45-µm filter and analyzed by HPLC. The morphology of micellar solution and lyophilized micelles reconstituted in water was observed by means of TEM.The stability of PTX/LAP micelles (lyophilized or micelle solution) was analyzed for the percentage of initial drug loading and morphology. Micelles were stored at 4 °C for 6 weeks. For drug quantitation, the micellar solution was centrifuged at 12,300× 2.MCF-7 human epithelial breast cancer cells were obtained from the American Type Culture Collection (ATCC). MCF-7 were cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented with 10% heat-inactivated fetal bovine serum , 10 mM HEPES buffer (Sigma-Aldrich), 100 U/mL penicillin, 100 μg/mL streptomycin (Sigma-Aldrich), and 0.01 mg/mL human recombinant insulin (Sigma-Aldrich). Cells were maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO4 cells/well and 96-well plates were used. The cultures were carried out for 24, 48, or 72 h. Control cells were incubated in the medium containing the non-toxic concentration of DMSO  or blank micelles in the same concentration as the experimental ones. At the end of the incubation period, the cytotoxicity was measured using a sulforhodamine assay according to the protocol described earlier [Free PTX and LAP stock solutions were prepared in DMSO and, as with the free micelles and drug-loaded micelles, were diluted with the appropriate volumes of the growth medium directly before the experiment. After 24 h of culture, the medium was replaced with the media containing PTX (25 nM), LAP (25 nM), the binary mixture of PTX and LAP, or PTX/LAP-loaded PLA-PEG micelles with the same concentration of the drugs. The initial cell density was 1 × 10 earlier . Absorba4 cells/well in 100 μL of complete medium, were cultured in 96-well flat clear-bottom black-wall microplates for 24 h. Then, the cells were treated with PTX (25 nM), LAP (25 nM) or the binary mixture of PTX (25 nM) and LAP (25 nM) in DMSO or PTX, LAP or PTX/LAP-loaded PLA-PEG micelles at the same concentration for 1 h. After pretreatment with the compounds, 100 μL of the prepared calcein-AM solution was added to each well followed by incubation at 37 °C for another 30 min in the dark. Intracellular fluorescence was measured with a microplate reader  with an excitation wavelength of 485 nm and an emission wavelength of 535 nm. P-gp activity was expressed as a percentage of fluorescence measured in the treated wells relative to that in the untreated control wells. P-gp activity was examined by determination of the intracellular accumulation of calcein, the fluorescent anionic dye, using the Multi-Drug Resistance Assay Kit  according to the manufacturer’s protocol. Calcein acetoxymethyl ester  is a lipophilic, nonfluorescent dye that diffuses into cells where it is cleaved by cytosolic esterase to a hydrophilic and intensely green fluorescent calcein, which is well retained in the cytosol . MCF-7 cMean values ± standard deviations were calculated from the data obtained in 4 independent series of experiments. Statistica 10 PL software for Windows  was used to perform variance analysis (ANOVA) and Tukey’s HSD test. The p-value of less than 0.05 was considered significant. PLA-PEG was used to prepare micelles for the co-delivery of PTX and LAP. As shown in 1H NMR spectra of individual drugs, lyophilized drug-free micelles and lyophilized drug-loaded micelles. Signals arising from the methine (5.2 ppm) and methyl (1.5–1.4 ppm) protons of PLA and methylene protons of PEG (3.6 ppm) were observed in the drug-free micelles. In the spectrum of PTX/LAP-loaded micelles, signals arising from the drugs were detected in addition to all the signals of the copolymer. PTX was identified by proton resonances at 1.0 ppm assigned to its methyl groups at C16 and C17 and LAP by signals at 8.6 and 6.5 ppm belonging to the aromatic groups. As shown in N-allylamino-17-demethoxygeldanamycin) [The effective incorporation of PTX and LAP into micelles was shown by means of NMR analysis. namycin) .The release of drugs from the micellar system was studied under in vitro conditions . Similart/M∞ = k1tm+ k2tm2 . The coefficient m is the purely Fickian diffusion exponent [2adjusted = 1.00 and R2adjusted = 0.99 for PTX and LAP, respectively). LAP was mostly released by Fickian diffusion (m = 0.42) [m for PTX (m = 7.62) indicates anomalous transport [In order to determine the mechanism of drug release from the micelles, the release data were fitted to Peppas–Sahlin kinetic model, which is described by the following expression: Mexponent . The Pepexponent . The rel = 0.42) . The higransport . The higransport ,27.co-PEG [To determine the stability of the drug-loaded micelles, the drug content and morphology was analyzed . Lyophilco-PEG . The PTXThe cytotoxic effects of the blank micelles, PTX-, LAP-, and PTX/LAP-loaded PLA-PEG micelles were tested in vitro on MCF-7 breast cancer cells. A comparison was made with a mixture of free drugs in the same concentrations as in the drug-loaded micelles. The MCF-7 cell line, derived from the mammary glands’ metastatic sites, was chosen as a model for the study because of the lack of EGFR and HER-2 receptors which implies its insensitivity to LAP. As shown in To find out whether free LAP and drug-loaded PLA-PEG micelles overcome MDR in MCF-7 breast cancer cells, calcein-AM assay was performed to determine their effect on P-gp activity. The kit contained a cell-permeable nonfluorescent P-gp substrate , which is cleaved intracellularly into a fluorescent molecule  for the detection of P-gp activity.As shown in It is known from several in vitro and in vivo studies that LAP not only effectively inhibits the proliferation of HER-2-positive breast cancer but also strongly sensitizes PTX-resistant tumors to PTX . Co-trea1H NMR. TEM analysis revealed the dual morphology of micelles, which is advantageous for providing a higher initial dose of the active agent by spherical micelles and the prolonged release by filomicelles. In fact, the micelles provided the release of PTX and LAP for over 264 h. The PTX/LAP-loaded PLA-PEG micelles (lyophilized and solution) showed high stability during storage at 4°C for at least 6 weeks. Drug-free micelles did not affect the viability of cells. Moreover, this study showed the feasibility of using a LAP and PTX combination to overcome MDR in MCF-7 cells. It is concluded that PTX/LAP micelles could be promising for uses not only in the therapy of HER-2-positive, but also HER-2-negative breast cancers.PLA-PEG bioresorbable micelles present many advantages as drug delivery systems, e.g., biocompatibility and sustained drug release. This study aimed to develop a novel micellar carrier for the co-delivery of PTX and LAP. The simultaneous incorporation of both drugs into PLA-PEG micelles was confirmed by


Eucommia ulmoides Oliv. leaf is a traditional Chinese antihypertensive and antidiabetic medicine. We examined the effects of chronic Eucommia leaf extract (ELE) administration on artery function and morphology in spontaneously hypertensive rats (SHRs). ELE was orally administered via normal diet ad libitum to six-week-old male SHRs at a concentration of 5% for seven weeks. Acetylcholine (ACh)-induced endothelium-dependent relaxation, sodium nitroprusside (SNP)-induced endothelium-independent relaxation, plasma nitric oxide (NO) levels, and media thickness were assessed. ELE significantly improved ACh-induced aortic endothelium-dependent relaxation but did not affect SNP-induced endothelium-independent relaxation in the SHRs, as compared to the animals receiving normal diet. Plasma NO levels and media thickness were significantly increased and decreased, respectively, in the ELE-treated SHRs. Therefore, long-term ELE administration may effectively improve vascular function by increasing plasma NO levels and bioavailability, and by preventing vascular hypertrophy in the SHR aorta. According to a World Health Organisation report, cardiovascular disease (CVD), including heart attacks, heart failure, kidney disease, and stroke, accounts for approximately 17 million deaths per year, corresponding to almost one-third of total global mortality ,3. There2 [The vascular endothelium plays an important role in the regulation of vascular tone, tissue blood flow, inflammatory responses, and maintenance of blood fluidity ,5. Vasod2 . NO is a2 . Endothe2 .Eucommia ulmoides Oliver is the only known species of the genus Eucommia, which is the only genus in the Eucommiaceae family. The bark of E. ulmoides (Cortex Eucommiae) is used to produce herbal medicines that are traditionally used as analeptic, analgesic, sedative, antihypertensive, and diuretic drugs [Eucommia leaves (ELs) have been used in the Sichuan District of China as an antihypertensive drug and a health food [ic drugs ; it is alth food . In Japain vivo and in vitro studies, indicating that ELE exhibited a dose-dependent blood-pressure-lowering effect in the spontaneously hypertensive rat (SHR) [in vitro [Previously, several pharmacological studies reported that EL extract (ELE) also exhibited antihypertensive , antihypat (SHR)  and elicin vitro ; long-tein vitro ,22. Howein vitro ,24. Chrop < 0.01 or 0.05) than those in the Wistar Kyoto rat (WKY) group. The ΔSBP in the SHR-ELE group was also significantly higher (p < 0.01) than that of the WKY group. However, the ΔSBP values in the SHR-ELE group at weeks three and seven were significantly lower (p < 0.01 or 0.05) than those in the SHR-control group  cumulatively to aortic tissues that had been pre-contracted with phenylephrine . −7–10−4 M ACh in the SHR-control group was significantly lower (p < 0.01) than that in the WKY group. The effective concentration causing 50% relaxation (EC50) in the SHR-control group was 0.61 ± 0.19 μM, which was significantly higher (p < 0.01) than that of the WKY group (0.076 ± 0.014 μM). In contrast, the ACh-induced relaxation response at 10−7.5–10−4 M in the SHR-ELE group was significantly higher (p < 0.01 or 0.05) than that in the SHR-control group. The EC50 of the SHR-ELE group was 0.15 ± 0.06 μM, which was significantly lower (p < 0.01) than that of the SHR-control group. 50 values did not differ significantly between the three groups . The NO level in the SHR-ELE group (11.75 ± 1.95 μM) was significantly increased compared with that in the SHR-control group.NO is produced from arginine by endothelial NO synthase (eNOS) in the vascular endothelium and is responsible for ACh-induced relaxation. Therefore, we examined the plasma NO levels to evaluate the improvement in endothelial function following ELE treatment . The plap < 0.05). The arterial media thickness was significantly decreased in the SHR-ELE group , as compared with that in the SHR-control group  , and oxiIn addition, the inhibitory effect of ELE treatment on vascular hypertrophy might relate to the increased NO level. SNP-induced relaxation of the thoracic aorta did not differ significantly among the three study groups, which suggested that their mesothelial function did not differ during the study period. However, the media was significantly thicker in the SHR-control group than in the SHR-ELE group. In addition to its vasorelaxant effects, NO has a pivotal role in suppressing the abnormal proliferation of VSMCs . Increasin vitro antioxidant activity [in vitro antioxidant activity [ELs contain compounds including the iridoid glucosides, geniposidic acid (GEA), and asperuloside (ASP); the caffeic acid derivative, chlorogenic acid (CHA) ,36. It hactivity  and has activity . Quercetactivity , howeverTo our knowledge, there are no reports of toxic or negative health effects of ELE or ELE-containing foods. In this study, each individual rat in the SHR-ELE group ingested 60.5 mg of GEA per day for seven weeks and we did not observe any sign of toxicity.In conclusion, to the best of our knowledge, this study is the first to demonstrate that long-term ingestion of ELE effectively improved the aortic endothelium- and NO-dependent relaxation, as well as increasing the plasma NO level, thereby decreasing vascular hypertrophy in SHRs. Therefore, ELE treatment may be effective in restoring vascular function.4), monobasic potassium phosphate (KH2PO4), potassium chloride (KCl), sodium chloride (NaCl), sodium hydrogen carbonate (NaHCO3), SNP, and xylene were purchased from Kanto Chemical Co., Inc. . ACh, 10% buffered formalin, PE, papaverine (PV), ethylenediaminetetraacetic acid (EDTA) 2K, and 30% hydrogen peroxide were purchased from Wako Pure Chemical Industries . Citraconic anhydride , 10% normal donkey serum, hematoxylin, and diaminobenzidine were purchased from Nisshin EM  and Abcam , Dojindo Laboratories , and Merck KGaA , respectively.Ethanol (EtOH), magnesium sulfate (MgSOn = 14) and WKY/Izm (n = 7) were purchased from Japan SLC, Inc. . They were housed in the Animal Research Center of the Faculty of Agriculture at Shinshu University under a controlled ambient temperature of 23 ± 1.0 °C, with 50% ± 5% relative humidity and a 12-h light/dark cycle. After an adaptation period of one week, six-week-old rats were used for the experiments. All animal experiments were carried out in strict accordance with the recommendations of the standards relating to the Care and Management of Laboratory Animals and Relief of Pain (2006) published by the Japanese Ministry of the Environment. The protocol was approved by the Animal Care Committee of the Faculty of Agriculture at Shinshu University (permit number: 250043). The surgeries were performed under diethyl ether anesthesia, and all efforts were made to minimize animal suffering and discomfort.Five-week-old male SHR/Izm (The ELs were collected from the Sichuan District of China. ELE was prepared using a previously described method . Brieflyw/w) ELE to the MF diet. One day before the experiment, the SHRs were divided into two groups  that were fed either the MF diet (SHR-control group) or the MF diet with 5% (w/w) ELE (SHR-ELE group). The WKY rats  were fed the MF diet (WKY group). The effects of ELE were determined during a seven-week ad libitum administration. Rat body weights and their food and water intakes were measured twice a week throughout the experimental period. SBP was measured using the tail cuff method with a Softron BP98A  at one day before (day-1), and three and seven weeks after, the beginning of the experiment.A moderate-fat (MF) diet  was used as the control diet. The MF diet consisted of water (7.8%), proteins (23.1%), lipids (5.1%), ash (5.8%), dietary fiber (2.8%), and nitrogen-free extract (55.3%). The test diet was prepared by adding 5%  was anesthetized with diethyl ether and subsequently exsanguinated via the abdominal aorta. The isolated thoracic aorta was cut into 2-mm rings and placed in cold Krebs–Henseleit buffer . The endothelium was removed from some of the rings by gently rubbing the intimal surface with a small pair of forceps, to investigate the involvement of endothelium-derived NO in the subsequent measurements. The rings were mounted at a resting tension of 1.5 g in a 5 mL organ bath  containing warmed (37 °C) and oxygenated  Krebs-Henseleit buffer. After a 60 min equilibration at this tension, vasoconstrictor PE (0.3 μM) was added to the rings. When a constriction plateau was reached, the removal of the endothelium was confirmed by applying 100 μM ACh to the constricted rings. After checking the endothelial condition, the tissues were constricted again with PE. Upon attaining a constriction plateau, endothelium-dependent and -independent relaxation was evaluated using the concentration-response curves for ACh (10−9–10−4 M) in the endothelium-intact rings and those of SNP (10−11–10−5 M) in the endothelium-denuded rings. The vasorelaxant effect was expressed as the percentage relaxation of PE-induced constriction. At the end of the evaluation of endothelial function using ACh, the rings were completely dilated by the addition of excess vasorelaxant (100 μM PV) to confirm that the mesothelium functioned properly.The endothelial function of the thoracic aorta of each rat was measured at the end of the experimental period. The assay was performed according to the method of Nakamura et al. . Brieflyg for 10 min at 4 °C  to separate the plasma. The total NO (nitrite and nitrate) in the plasma was determined using the Griess reaction. A nitrate/nitrite colorimetric assay kit, where nitrate was converted to nitrite by nitrate reductase, was employed according to the manufacturer’s instructions . The measurements were performed in triplicate and the nitrate/nitrite concentrations (μM) were expressed as the means ± standard error (SE).The blood samples collected during exsanguination were transferred to a tube containing 10% EDTA-2K anticoagulant. The uncoagulated blood was centrifuged at 1811× To visualize the smooth muscle cells of the aorta, the sections were stained using an avidin-biotin-peroxidase complex method. Briefly, the thoracic aorta of each rat was isolated as described in t-test for unpaired observations. p < 0.05 was considered statistically significant.All data were expressed as the means ± SE. The statistical significance of differences between two groups was estimated using Student’s The results of this study demonstrate that chronic administration of ELE effectively restored the aortic endothelium-dependent, NO-mediated relaxation, and the plasma NO level, which decreased arterial media thickening in SHRs. These data suggest that ELE may exert anti-endothelial dysfunction and anti-vascular hypertrophy effects.


P < 0.05), while there was an elevation on the p62 level. ET appreciably mitigated the OVX-induced negative effects on muscle quality and the autophagy pathway, which seemed to be dependent on ET volume. The most optimal outcomes were observed in the OVX-90ET group. The OVX-120 group had an adversely augmented catabolic process associated with gastrocnemius muscle atrophy. In conclusion, the expression levels of autophagy proteins are decreased in OVX rats, which can be appreciably mitigated following 8 weeks of swimming ET.Ovariectomy disrupts estrogen production and homeostasis. However, whether exercise training (ET) could counteract the ovariectomy-induced effect on muscular autophagy has remained elusive. This study examined muscular autophagy in ovariectomized (OVX) rats following 8 weeks of swimming ET. Here, 40 6-month-old female Sprague-Dawley rats were randomly divided into five groups: sham-operated control (Sham), OVX control (OVX), OVX with 60-min ET (OVX-60ET), 90-min ET (OVX-90ET), and 120-min ET (OVX-120ET) for 6 days/week. According to the results of Western blotting, the expression levels of autophagy-related proteins in the OVX gastrocnemius muscle, including mammalian target of rapamycin, uncoordinated 51-like kinase 1, Beclin-1, autophagy-related gene (Atg-7), and microtubule-associated protein light chains 3 were significantly decreased (all  Declination of ovarian function in aging women is accompanied by decreasing estrogen. Studies have reported that postmenopausal women are more susceptible to having obesity, cardiometabolic diseases, osteoporosis, and sarcopenia . SubsequSkeletal muscles represent 40–45% of body mass in a healthy, lean individual  and funcIn initiating autophagy process of skeletal muscles, the mechanistic  target of the rapamycin (mTOR) is presented as a key regulator that inhibits autophagy initiation  while itIt has been previously reported in a rodent model that wheel-running exercise for 4 weeks promoted protein expression of autophagy and increased autophagy flux in the plantaris muscle , and treHere, 40 6-month-old female Sprague-Dawley rats (180–220 g) were provided by the Center of Experimental Animal, Xuzhou Medical University . All rats were housed under a humidity (50 ± 10%) and temperature controlled (24 ± 1°C) room with 12:12 h light–dark cycle. All animals were allowed freely to access water and rodent chow throughout the study. The study protocol and experiment (Protocol #2016-036) were reviewed and approved by the Animal Ethics Committee of Xuzhou Medical University in accordance with the Guidelines for Ethical Conduct in the Care and Use of Animals.n = 8). The incisions were sutured and dressed with povidone iodine for 7 days (n = 8 for each): OVX group with no intervention (n = 8) and OVX rats which underwent ET for 60 min/day , 90 min/day , and 120 min/day .After a week of acclimatization, 40 rats were subjected to a bilateral ovariectomy operation. Under anesthesia through intraperitoneal injection with 10% chloral hydrate (3.5 ml/kg), an incision of about 1.2 cm was made in each of the dorsal flank regions of the rat to expose the ovaries. The fallopian tubes were sutured at the most distal ends and the ovaries were excised for 32 rats. The other eight rats were subjected to the same surgical procedure without excising ovaries to serve as the Sham group  . Each spBlood samples were collected and immediately chilled on ice in tubes containing 5 μl heparin. The samples were centrifuged at 4000 rpm for 15 min at 4°C and then stored at -80°C until analysis. Serum estrogen concentrations were determined using an E2 enzyme-linked immunosorbent kit  .The gastrocnemius skeletal muscle tissues were homogenized in a lysis buffer  with a polytron homogenizer and an ultrasonic processor on ice. The bicinchoninic acid (BCA) assay  was used according to the manufacturer’s instructions to quantify the level of protein in each sample to ensure equal protein loading. The protein samples were denatured with sodium dodecyl sulfate (SDS) loading buffer (5×) and separated by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). Furthermore, 40 mg proteins along with a molecular weight protein marker  were subjected to SDS–PAGE using 7.5% acrylamide gel and electroblotted onto polyvinylidene fluoride (PVDF) membranes. The membrane was blocked with 5% non-fat milk in TBS containing 0.1% Tween 20 (TBS-T) and then probed at 4°C for 12 h with the primary antibodies against Beclin-1 , ULK1 , Atg-7, mTOR, LC3, and p62/SQSTM1 , and β-actin . After washing three times for 10 min with 0.1% TBS-T solution, the horseradish peroxidase (HRP)-conjugated secondary antibody (dilution of 1:4000 for goat anti-rabbit and dilution of 1:4000 for goat antimouse) was incubated at room temperature for 2 h, and signals were detected using Odyssey . Band intensities were analyzed using Image J 1.25 software  and the expression levels of all proteins were normalized to β-actin as described previously .t-test was applied to assess significant difference in body mass before and after the intervention. Two-way analysis of variance (ANOVA) was applied to test the significance of OVX factor and ET factor. Post hoc analysis with Tukey’s test was performed if the main factor had a significant influence. Statistical analyses were undertaken using SPSS 17.0 software . All data were reported as mean ± standard error of the mean (SEM). Statistical significance was accepted at P ≤ 0.05.A paired P < 0.01) of that was in the Sham group, despite the fact that all animals accessed food and water freely without restriction. ET seemed to have a bifurcated effect on body mass. The OVX-60ET (P < 0.05) and OVX-90ET (P < 0.01) groups gained more weight compared with the Sham group, whereas the OVX-120ET group had no difference in the change of body weight compared with the Sham group. Furthermore, body weight of the OVX-120ET group was significantly lower compared with the OVX, OVX-60ET, and OVX-90ET groups.Baseline body mass (prior to ET) was not statistically different among the groups . The ShaP < 0.01) lower after ovariectomy compared with the Sham group (P < 0.05) in estradiol level between the OVX and OVX-90ET groups. Ovariectomy resulted in a significant reduction in uterus weight . Nonetheless, the gastrocnemius muscle mass of the OVX120ET group was significantly lower compared with the OVX-60ET and OVX-90ET groups . These dAutophagy is essential for maintaining muscle mass and quality, as well as preventing muscle atrophy and dysfunction . CompareAutophagy initiation is controlled through activation of the ULK1 complex and is associated with the inhibitory influence of mTOR. Although there was a significant decrease in the intracellular level of mTOR observed in the OVX group, the expression levels of ULK and the downstream proteins in muscular autophagy cascade, including Beclin-1, LC3 and ATG-7, were all significantly decreased in the OVX group . These dSequestosome 1 (SQSTEM1) or p62 is an important cargo receptor involved in the autophagy lysosomal pathway and ubiquitin-proteasome system . The intHere, 8-week swimming ET effectively counteracted the OVX-induced suppressions of autophagy proteins. The intracellular levels of Beclin-1, Atg-7, and p62 of the gastrocnemius muscle in all training groups, i.e., OVX-60ET, OVX-90ET, and OVX-120ET, were not notably different compared with these proteins in the Sham group. Moreover, there was no significant difference in the expression level of mTOR of the gastrocnemius muscle among the OVX-90ET, OVX-120ET, and Sham groups. These data suggested that ET could be applied to mitigate the inhibitory influence (due to lack of estrogen hormones) on the autophagy pathway. In addition, this effect seemed to be training volume-dependent.Making a comparison among three training regimens with different exercise durations, i.e., OVX-60ET, OVX-90ET, and OVX-120ET, it was revealed that the 90-min training session was the most optimal. In the present study, the expression levels of mTOR, ULK1, and LC3 were lower in the OVX-60ET group compared with the Sham group, although they showed a trend of recovery. However, the expression levels of these proteins were not statistically different between the OVX-90ET and Sham groups. Although the OVX120ET group had similar intracellular levels of all these proteins compared with the OVX-90ET or Sham group, the p62 level of the OVX-120ET was appreciably lower than that in the OVX group. This observation indicated that the high training volume of the OVX120ET group could augment the autophagy–lysosomal pathway and catabolic process in the animals following ovariectomy, which consequently led to the atrophy observed in the gastrocnemius muscle . A previA major limitation of the study is that there was no quantitative histological assay for the gastrocnemius muscles in the animals. Differences in lean body mass and/or fat mass with and without estrogens between swimming and running ET should be further studied. Autophagy is critical in maintaining muscle mass and quality. Deficiency of estrogen in animals following ovariectomy shows a suppressed muscular autophagy pathway, which may lead to a disruption of intracellular homeostasis involved in the activations of the catabolic process and anabolic process. This may be a mechanism for the OVX animals, resulting in the increased fat infiltration/accumulation in the skeletal muscle. This mechanism explains why aging women have declined muscle quality or strength and increased frailty or falling incidence. Swimming ET with moderate exercise volume helps counteract ovariectomy-induced suppression on the muscular autophagy process and mitigates fat accumulation in the muscle. However, high training volume can adversely augment muscular autophagy and cause muscle atrophy.In conclusion, the expression levels of autophagy proteins are decreased in OVX rats, which can be appreciably mitigated following 8 weeks of swimming ET.The raw data supporting the conclusions of this manuscript will be made available by the authors, without undue reservation, to any qualified researcher.The study protocols and experiments were reviewed and approved by the Animal Ethics Committee of Xuzhou Medical University, which conformed to the Guidelines for Ethical Conduct in the Care and Use of Animals (Protocol #2016-036).WZ contributed to the hypothesis development, data collection, analysis and interpretation, and manuscript preparation. XS contributed to data interpretation and manuscript preparation. HY, HB, and LW contributed to the training of the rats and the data collection. RW contributed to the hypothesis development, data analysis and interpretation, and manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Huntington's disease (HD) is an inherited devastating neurodegenerative disease with no known cure to date. Several therapeutic treatments for HD are in development, but their safety, tolerability and efficacy need to be tested before translation to bedside. The monogenetic nature of this disorder has enabled the generation of transgenic animal models carrying a mutant huntingtin (mHTT) gene causing HD. A large animal model reflecting disease progression in humans would be beneficial for testing the potential therapeutic approaches. Progression of the motor, cognitive and behavioral phenotype was monitored in transgenic Huntington's disease minipigs (TgHD) expressing the N-terminal part of human mHTT. New tests were established to investigate physical activity by telemetry, and to explore the stress-induced behavioral and cognitive changes in minipigs. The longitudinal study revealed significant differences between 6- to 8-year-old TgHD animals and their wild-type (WT) controls in a majority of the tests. The telemetric study showed increased physical activity of 4.6- to 6.5-year-old TgHD boars compared to their WT counterparts during the lunch period as well as in the afternoon. Our phenotypic study indicates progression in adult TgHD minipigs and therefore this model could be suitable for longstanding preclinical studies of HD.This article has an associated First Person interview with the first author of the paper. Summary: The transgenic minipig model of Huntington's disease demonstrates a slow-progressing motor, cognitive and behavioral phenotype with later onset in adulthood. The clinical symptoms of neurodegenerative Huntington's disease (HD) are motor, cognitive and behavioral impairments manifesting typically in the mid-30s . Motor pIn 2009, a transgenic minipig model for HD (TgHD) expressing the N-terminal part of human mutated huntingtin  was generated . Generaln=8) and their WT controls (n=10) were monitored using motor, cognitive and behavioral tests; and, simultaneously, the physical activity of TgHD boars (n=6) and their WT siblings (n=6) at 2.5-6.5 years was investigated by telemetry.This study aimed to longitudinally monitor the motor, cognitive and behavioral phenotype of TgHD minipigs up to 8 years. Gait, hurdle and startbox back-and-forth tests using TgHD minipigs were established in the George-Huntington-Institute in Münster . ActivitP=0.013), and in TgHD sows at 4-5.9 years (P=0.015), compared with their age-matched WT controls. More evident changes in walking were observed among TgHD boars. TgHD animals exhibited different movement of the hind legs (Movie 1). Significant sex-related differences were observed between TgHD boars and TgHD sows at younger (4-5.9 years) and older (6-7.9 years) age .As the clearest clinical hallmark of HD is movement detriment, unsteady gait and lack of coordination in patients , the gaiAt the age of 4-5.9 years, both TgHD boars and TgHD sows had lower scores in the hurdle test B than thMovie 2) was introduced in minipigs. This test simulated unexpected disruption of an animal's balance. When 6-year-old TgHD boars were shoved in the haunch area, they lost their balance, and their hind legs slipped and moved in the opposite direction to which the sudden pulse occurred. Their front legs reached a wider position in an effort to maintain stability and not fall. Control WT animals quickly regained balance with no problems concerning their legs.Corrective responses to externally generated force pulses are disturbed in HD patients ; therefoP=0.004) decreased score in TgHD boars, but very similar score in TgHD sows, in comparison to their WT controls. However, the deepest-reached-hole test  lower score was observed in 6- to 7.9-year-old TgHD boars than in their age-matched WT controls, whereas TgHD females showed similar results to WT females. Similarly, 6- to 7.9-year-old TgHD males obtained a lower score than their WT counterparts in the cover pan test . TgHD animals were not willing to walk on the balance beam (Movie 3). TgHD sows showed significant age-dependent impairment in performance in the seesaw test (P=0.024).HD patients have difficulties in coping with stressful situations. Therefore, tests inducing stress were investigated; namely, a balance beam test and a seesaw test. The balance beam test A was preP=0.026) increased in comparison to that of WT controls. The physical activity of 4.6- to 6.5-year-old TgHD animals was also significantly higher (P=0.003) than that of 2.6- to 4.5-year-old TgHD animals. Similarly, in the afternoon period  increased with age.People with HD tend to be less physically active in the morning and more active in the afternoon and evening. This might be caused by sleep disturbances and shifted circadian rhythm in HD individuals . The phyn period C, physicTransgenic minipigs encoding truncated human mHTT  were monIn this study, walking, hurdle, pull-back, tongue, skittles, cover pan, balance beam, seesaw and physical activity tests were performed in a larger group of animals at the age of 4-5.9 and 6-7.9 years, and the impact of sex was also taken into account. An overview of the test outcomes is provided in Stricken gait problems to maintain upright posture and impaired balance are obvious motor symptoms in HD patients . ContrarConcerning fine motor skills, glossomotography revealed impairment of tongue protrusion in HD patients . In the Similar results were observed in skittle and cover pan tests. These tests were assessed to investigate cognitive decline . Age-depBalance beam and seesaw tests mirror response to stress stimuli and recollection of memories, representing the tied connection between cognition and emotions . Stress-Perspicuous sex-related differences were observed in all tests performed in this study. Although TgHD boars and TgHD sows both had reduced performance in the tests compared to their WT counterparts, TgHD boars showed a significant decrease in performance in the walking, balance, seesaw and skittles tests at the age of 6-7.9 years, but sows did not. The boars demonstrated more courage to complete the tasks, whereas the sows were generally fatter and more hesitant by nature, presumably leading to the results achieved. Sex commonly modulates human behavior, including many aspects of human brain development . The patTelemetric evaluation of physical activity revealed that TgHD boars of younger ages (2.6-4.5 years) had lower activity throughout the day than their WT controls, although this difference was non-significant. However, from the age of 4.6 years, physical activity was reduced in the morning period, and increased during the lunch and afternoon periods. A significant increase in physical activity was observed in TgHD boars with age, which could be associated with the manifestation of disease progression. Similarly, decreased morning activity and increased physical activity in the later hours of the day have been observed in HD patients. This feature relates to their impaired circadian organization .Recently, a new HD knock-in porcine model with 150 glutamines in the polyQ tract was generated using a CRISPR/Cas9 approach . WalkingIn conclusion, our longitudinal study has shown gradual progression in TgHD minipigs. The test outcomes correlate with disease progression and could be applied in preclinical tests, particularly in ongoing longitudinal AAV5-miHTT (microRNA targeting HTT sequence) studies in minipigs after they reach the age of phenotype manifestation. Based on promising results from the first minipig studies , the FooTable S1). Five animals died within the study . Six TgHD minipigs and their six WT siblings at the age of 2.6-6.5 years were used for the telemetry study (Table S1). See Table S1 for further details on the animals used. All experiments were carried out according to the guidelines for the care and use of experimental animals and approved by the Resort Professional Commission of the Czech Academy of Sciences for Approval of Projects of Experiments on Animals (approved protocol no. 53/2015).Transgenic minipigs  expressing one copy of N-truncated human mutated huntingtin with 124 glutamines in polyQ sequence  and theiChanges in gait were observed by walking the animal on a dry, flat floor. Gait was video-recorded at 6-month intervals. Scoring was as follows: 5 points for no visible gait problem and fluent walking; 4 points for slightly uneven weight bearing on one or more legs; 3 points for obvious deviation in weight bearing on one or more legs, with clear difficulties in walking; 2 points for lowering of the hind quarters close to the ground, placement of hind legs under the body; 1 point if the pig is unable to move; 0 points if the animal refused to perform the test .In the hurdle test A, the anThe animals were allowed to walk on a dry, flat floor and an unexpected shove was performed to the haunch area. Stability and reaction to the sudden external force were evaluated.The tongue test B was perIn the skittles test C, the anIn the cover pan test D, treatsThe balance beam test E, consisDuring the seesaw test F, the piA battery of motor, cognitive and behavioral tests was conducted three to five times per year. The mean data from all the measurements collected within 1 year were calculated for each individual animal. The groups of animals were selected according to their genotype, sex and age. Each data point in the graph represents one individual animal at a defined age. Individual animals were tested within the 4 subsequent years, thus data were collected from their ages at x, x+1, x+2 and x+3 years.TgHD boars were monitored for 1 week in each session. Physical activity was evaluated over three periods: morning , lunch  and afternoon . Telemetric system rodentPACK2 obtained from emka TECHNOLOGIES (France) was used in the experiments. The telemetric system consisted of small transmitters and central receivers. Every transmitter could acquire x, y, z and total acceleration. Data were sampled at a frequency of 100 Hz with resolution ±2 g. Minipig boars wore collars with transmitters during the experiments . A PC coPhysical activity was measured in six sessions . The mean data from all measurements collected within 1 year were calculated for each individual animal. The groups of animals were selected according to their genotype and age. Each data point in the graph represents one individual animal at a defined age. Individual animals were tested within the 4 subsequent years, thus the data were collected from their ages at x, x+1, x+2 and x+3 years.t-test and Mann–Whitney test. All figures show the mean results and error bars represent s.e.m. Significance was set at P≤0.05 .Evaluators of all of the tests were blinded for animal genotype. All statistical tests were carried out in GraphPad Prism. Calculation of statistical significance was computed using Student's http://dmm.biologists.org/collection/neuromuscular.This article is part of a special collection ‘A Guide to Using Neuromuscular Disease Models for Basic and Preclinical Studies’, which was launched in a dedicated issue guest edited by Annemieke Aartsma-Rus, Maaike van Putten and James Dowling. See related articles in this collection at


The development of a sacrificial bond provided unique inspiration for the design of advanced elastomers with excellent mechanical properties, but it is still a huge challenge to construct a homogenous polar sacrificial network in a nonpolar elastomer. In this effort, we proposed a novel strategy to engineer a multi-ionic network into a covalently cross-linked 1,2-polybutadiene  facilitated by in-situ intercalated organic montmorillonite (OMMT) without phase separation. XRD, SEM, and TEM analysis were carried out to characterize the microstructure of the resulting polymers. Crosslinking density, dielectric performance, and cyclic tensile tests were used to demonstrate the interaction of zinc methacrylate (ZDMA) and OMMT. The dynamic nature of ionic bonds allowed it to rupture and reform to dissipate energy efficiently. Stretching orientation brought parallelism between polymer chains and OMMT layers which was beneficial for the reconstruction of the ionic network, ultimately resulting in high strength and a low stress relaxation rate. Overall, our work presented the design of a uniform and strong sacrificial network in the nano-clay/elastomer nanocomposite with outstanding mechanical performances under both static and dynamic conditions. The nacreous layer in biological materials—byssus in mollusks and bones in mammals—provide a paradigm for a stiff, strong, and at the same time tough protective engineering ,2. The e2Zn ion pairs can graft onto rubber molecular chains through a peroxide-induced vulcanization system [Atactic 1,2-polybutadiene  rubber with dense vinyl groups and few double bonds on backbone, exhibiting excellent wet-skid resistance, low rolling resistance, low heat build-up, and superior aging resistance, can meet the requirements of high-performance green tire when used in combination with other rubbers such as natural rubber ,30,31,32n system ,37.In this work, we incorporated ZDMA-induced ionic sacrificial bond into an in-situ intercalated OMMT/1,2-PB nanocomposite (in-situ-NC) to reinforce the covalently cross-linked elastomer by the interaction of ionic bond and nano-clay. The OMMT layers mostly exfoliated by in-situ polymerization served as bricks and the ionic bonds which could rupture before covalent bond acted as mortar to enhance the strength of the resulting polymer. As expected, such reinforcing engineering brought excellent properties to 1,2-PB.3), ethyl alcohol, and hydrochloric acid were purchased from Tianjin Bodi Chemical Co., Ltd., Tianjin, China. The main catalyst (Mo) and co-catalyst (Al) for coordination polymerization were prepared as reported in our previous work [1,3-Butadiene (Bd) and n-hexane were offered by Qilu Petrochemical Corp., Zibo, China. 1,3-Butadiene was dried over activated aluminum oxide and distilled twice prior to use. n-Hexane was distilled with 67–70 °C fraction for later use. Na-MMT with the cation exchange capacity (CEC) of 60~70 mmol per 100 g was supplied by Zhejiang Fenghong Clay Chemical Co., Huzhou, China. Cetyl trimethyl ammonium bromide (CTAB), silver nitrate (AgNOous work . Nitroge3 with a concentration of 0.1 M. Then the product was dried under vacuum at 80 °C to constant weight and ground to powder.MMT (20 g) was dispersed into 1 L deionized water, heated to 80 °C and stirred for 2 h. Thereafter 18 mmol of CTAB in water solution was slowly added to the MMT suspension and stirred for 3 h with the PH value being adjusted to 3 to 5. After the reaction was finished, the suspension was filtered with deionized water, until there was no bromide ion detected by AgNO−3) and co-catalyst (Al/Mo by a molar ratio of 30) were subsequently injected into the reactor to react for 6 h. Then the polymerization was terminated by ethyl alcohol. After the termination of the polymerization, the resulting nanocomposite was ultimately poured into the boiling water to precipitate and remove most of the main catalyst and co-catalyst. Then the precipitates were dried in vacuum at 80 °C to a constant weight. The resulting in-situ intercalated OMMT/1,2-PB nanocomposite was named as in-situ-NC.Under a nitrogen atmosphere, a certain amount of OMMT and Bd solution in n-hexane (with a concentration of 2.6 M) were firstly added into a reactor and heated to 60 °C. Then, the main catalyst . The resulting physical blended OMMT/1,2-PB nanocomposite was named as ph-NC.In-situ-NC or ph-NC was compounded with ZDMA in a two-roll mill, respectively. Then the samples were subjected to compression molding at 135 °C. The formulations are listed in α (λ = 0.154 nm) was used as an X-ray source with a generator voltage of 40 kV and a current of 100 mA.XRD patterns were carried out using a Rigaku D/max RB X-ray diffractometer  with the diffraction angles from 1° to 10°. CuKThe molecular weight and molecular weight distribution of 1,2-PB and the in-situ-NC were characterized with a Waters Co. Maxims 1515 GPC instrument . The GPC measurements were conducted by injecting 100 μL sample solution at a flow rate of 1.0 mL/min at room temperature with tetrahydrofuran as the leaching solvent. The specimen concentration was 1 mg/mL.−1 by total reflection mode.FTIR was obtained on a Nicolet FTIR-Magna-750 spectrophotometer  in the range of 4000–400 cm1H spectra of NMR were measured on a Bruker Ultra-Shield™ spectrometer  at 500 MHz with deuterated chloroform as the solvent and tetramethylsilane as an internal chemical shift reference.SEM analysis was performed on a JMS-6700F  scanning microscope equipped with an energy-dispersive X-ray  microanalytical system. The samples were cryo-fractured in liquid nitrogen prior to SEM testing.The EDS spectra and EDS maps were carried out by energy dispersion spectroscopy .For the observation of transmission electron microscopy (TEM), the specimens were ultramicrotomed into thin pieces of ~100 nm in thickness with UC7-532319  microtome under a liquid nitrogen atmosphere. Then, the observations were carried out on a JEM-3010  transmission electron microscope at an acceleration voltage of 100 KV.rΦ is the volume fraction of the rubber in the swollen mass, V0 is the molar volume of the toluene which is 106.2 cm3, n is the crosslinking density, and χ is the Flory-Huggins polymer-solvent interaction term, which is 0.393 for toluene [rΦ was obtained according to Bala et al. [m1 and m2 are the masses of the swollen sample before and after dried, respectively. ρ1 and ρ2 are the densities of toluene (ρ1 = 0.865 g/cm3) and rubber, respectively.Samples were immersed in toluene at room temperature for 72 h to achieve their swollen equilibrium. Then, the swollen samples were blotted with tissue paper to remove the excess toluene and immediately weighed. Finally, the samples were dried at 80 °C to constant weight. The crosslinking density was determined from the Flory–Rehner equation :(1)−[ln toluene . The Φr a et al. (2)Φr=m2Tg) was conducted using a TA DSC-Q20 thermal analyzer, Milford, MA, USA, over a temperature range from −80 to 40 °C at a heating rate of 10 °C/min.Glass transition temperature  under temperature sweeping mode from −60 to 80 °C at a heating rate of 3 °C/min, an amplitude of 10 µm and a frequency of 1 Hz.Under different temperatures , samples were quickly stretched to 50% and maintained for 10 min. Then the stress was reduced to zero, and the residual deformation was measured for 10 min.The loading-unloading cycles were performed on a DMA Q800  instrument with an extension rate of 50%/min at room temperature. In each cycle, the sample was stretched to 100% strain. After the second cycle, the sample was heated at 80 °C for 10 min and then cooled at 25 °C for 10 min to heal the dynamic network, followed by another loading–unloading cycle.7 to 10−2 Hz with a voltage of 1 V at room temperature. The samples were subjected to gold sputtering for 100 s before testing.Dielectric performance was performed on an Alpha-A  novocontrol under frequency sweeping mode from 10Tensile and tear strength were tested on a GT-AT-7000M  electronic tension testing machine at a speed of 500 mm/min. Hardness test was performed on a shore A hardness test instrument according to ISO 7619-1:2010. DIN abrasion test was performed on a GT-7012-D abrasion testing machine according to ISO 4649-2010.dsinθ = n λ). After the organic modification, the diffraction angle of OMMT was 2.21°, indicating that the interlayer spacing was enlarged to 4.01 nm. Then the OMMT was in-situ intercalated with Bd via Mo-catalyzed coordination polymerization. The relationship between the conversion rate of Bd monomer and the mass fraction of OMMT was shown in Organically modified montmorillonite (OMMT) with lower surface energy and larger interplanar spacing exhibits better dispersity in organic materials than pure montmorillonite (Na-MMT). XRD patterns of Na-MMT and OMMT were shown in The degree of polymerization and molecular weight distribution were determined by gel permeation chromatography GPC, . The degδ = 4.9, δ = 5.3 and δ = 5.5 ppm were attributed to the olefinic protons of =CH2 of 1,2-unit, -CH= of 1,4-unit and -CH= of 1,2-unit, respectively. The 1,2-unit content (C) was calculated according to the following equation:A was the integral area of the corresponding signal peak. After in-situ intercalated with OMMT, the 1,2-unit content of the NC was 83% which was the same as that of unmodified 1,2-PB. The results of FTIR and 1H NMR showed that, although the negative ions on the surface of OMMT may devitalize the Mo catalyst, it couldn’t change the structure of active centers in this coordinative polymerization procedure.The 1,2-unit content of 1,2-PB and in-situ-NC was calculated by NMR spectra . The sig2Zn groups into rubber matrix. Then the Zinc ion (Zn2+) was able to joint with the negative ion on OMMT to form OMMT-polymer ionic bonding or with the negative ion on an adjacent polymer to form inter-chain and intra-chain ionic bonding. Additionally, -(COO)2Zn groups could aggregate together to create a small ion cluster. Therefore, OMMT-polymer ionic bonding, inter-chain, and intra-chain ionic bonding together with ionic cluster constituted a multi-ionic supramolecular network –O conjugated with C=C double bonds. The relative intensity of in-situ-NC/ZDMA40, after curing, was obviously weak at 1244 cm−1 due to the reaction of C=C double bonds. Moreover, more ZDMA was grafted onto 1,2-PB molecular chains for in-situ-NC according to the weaker peak intensity at 1244 cm−1. The main reason was that the negative ions on the surface of OMMT layers led to a better dispersity of ZDMA.Moreover, the exfoliated OMMT layers would leave a more exposed Si–O bond. Additionally, the formation of a covalent linkage between the ZDMA and the double bond on 1,2-PB was illustrated by FTIR analysis b. In theHowever, the crosslinking of 1,2-PB double bonds and the grafting of ZDMA is a pair of competitive reactions. In this work, we controlled the crosslinking density of covalent and ionic networks by adjusting reaction temperature and time. The curing curves of the in-situ-NC/ZDMA40 under different temperatures were shown in −4 mol/cm3 was significantly lower than that of in-situ-NC/ZDMAs due to the non-exfoliated OMMT. Under the same curing condition, the covalent crosslinking density for in-situ-NC was almost zero and for in-situ-NC/ZDMAs was more than 0.8 × 10−4 mol/cm3. With the increasing mass fraction of ZDMA, both ionic and covalent crosslinking densities for in-situ-NC/ZDMAs increased at first and then decreased. The increasing trend illustrated that ZDMA not only determined the formation of the ionic network but also affected the generation of covalent bond for in-situ-NC/ZDMA because the -(COO)2Zn groups on ZDMA might accelerate the decomposition of DCP. Therefore, the covalent crosslinking density increased significantly for ph-NC/ZDMA40 and in-situ-NC/ZDMAs. However, too much ZDMA in in-situ-NC/ZDMAs is likely to agglomerate into particles which embed DCP and thereby results in the homopolymerization of ZDMA to form poly-ZDMA (PZDMA). Accordingly, the reaction rate for the grafting of ZDMA and the crosslinking of 1,2-PB decreased for in-situ-NC/ZDMA40. Additionally, the total crosslinking density could be further confirmed by the torque value in a curing curve  can be described as follows [7 ergs/K mol), and ρ is the density of the polymer. Thus the total crosslinking density could be calculated by DMA and the results were shown in DMA testing typically shows a small sample deformation during modulus measurements. In this way, the sample can respond to deformation by changing the chain conformation. When the deformation is accommodated by conformational changes, without bending or breaking of bonds, the relationship between rubbery plateau modulus and crosslinking density ( follows :Ve = E′/Tg) determined by DSC  . Accordid by DSC  also shoTg. The orientation of OMMT layers and polymer chains were in the same direction, which was advantaged for the reconstruction of the ionic network. Meanwhile, the increase in the maximum stress displayed the formation of a denser ionic network induced by the orientation mentioned above. After heating at 80 °C, the orientation of the covalent network of in-situ-NC and ph-NC/ZDMA40 nearly completed self-recovery, and its cyclic tensile curve almost overlapped that of the first cycle. However, in-situ-NC/ZDMA40 showed smaller stress during loading after heating than that of the first cycle before heating. It seems to form a weaker ionic network after heating, which, combining with the denser network by stretching, indicated that the ionic network is sensitive to the conformation of polymer chains. In addition, the samples with other content of ZDMA exhibited similar hysteresis behaviors (Sacrificial bond is a general conception of weaker bond in polymer and it is difficult to prove its sacrifice directly. Rief et al. reported that the excellent strength and toughness in natural materials could be characterized by a saw-tooth pattern observed from single-molecule force–extension curves . The sawehaviors .To further illustrate the reinforcing effect of ionic crosslinks, stress relaxation analysis, was performed at 25 °C a. All thAfter the relaxation experiment and recovery for 10 min, the residual deformation was measured and was shown in 2Zn groups. Nevertheless, the mechanical properties of in-situ-NC/ZDMA40 decreased attributed to the large ZDMA aggregates acting as impurities. Meanwhile, the abrasion resistance of in-situ-NC and in-situ-NC/ZDMAs decreased with the increasing fraction of ZDMA owing to the fact that the ionic contacts decreased the flexibility of the polymer chains. Moreover, the abrasion loss of ph-NC/ZDMA40 was also very high, resulting from the weak interaction of the polymer chain and ZDMA aggregates.The mechanical properties of in-situ-NC, in-situ-NC/ZDMAs and ph-NC/ZDMA40 were listed in We developed a nano-clay/elastomer nanocomposite with high tensile strength, tearing strength and low stress relaxation rate which was reinforced by a multi-ionic supramolecular network including OMMT-polymer ionic crosslink, inter-chain, or intra-chain ionic crosslink as well as small ion cluster. The exfoliated OMMT layers prevented the aggregation of ZDMA in nonpolar rubber matrix through ionic interaction between OMMT and ZDMA, and thereby resulted in a uniform ionic network. The ionic network could rupture preferentially during stretching to protect the covalent network and dissipate more energy. Additionally, the ionic crosslinks could be reconstructed and be strengthened through the orientation of OMMT and polymer chain. However, the reversibility of such ionic bonds enlarged the residual deformation of the resulting polymer. In future studies, we will further improve the ionic crosslinking density and construct a stronger sacrificial network to prepare shape memory or self-recovery materials and study the dynamics of ionic crosslinking.


S. typhimurium and E. coli O157:H7. The CF-CAR had higher antioxidant capacity and an increased protective effect against oxidation of erythrocytes in different grades. These results suggest potential applications of CF-CAR as active packaging to preserve food products.Chitosan films (CF) with carvacrol (CAR)  were prepared by the emulsion method. The retained CAR, water solubility, water vapor permeability (WVP), optical, mechanical properties, antibacterial and antioxidant capacity of films were analyzed. The results indicate that the retention of CAR in the CF was ≈50%. The incorporation of CAR to CF decreased the water solubility, the WVP, the yellowing and transparency and the tensile strength, but increased the stiffness. Microcapsules with diameters of 2 to 7 µm were found on the surface CF-CAR. The CF-CAR with highest CAR concentrations showed antibacterial activity against  Currently, consumers demand foods with low levels of synthetic chemical preservatives or even free of them . This phd-glucosamine and β-(1-4)-N-acetyl-d-glucosamine, obtained by alkaline deacetylation of chitin. This viscous polymer is biodegradable, nontoxic, antigenic, antimicrobial and biocompatible , compared to the 1.5% CF-CAR [43.1% (0.136 ± 0.0010 g/g TWF)] ( < 0.05) . The los  was evaluated. Salmonella showed sensitivity against CF-CAR at 1.0% and 1.5%  and E. coli (1 mmol/L)  was determined by the method of Lu et al.  with theThe experimental data was subjected to analysis of variance (ANOVA) using the NCSS Statistical Analysis System . Results were expressed as the mean value ± standard deviation (SD). The analysis of variance and significant differences were analyzed by Duncan’s multiple range tests at a 0.95 confidence level.A considerable amount of CAR was present (≈50%) in the CF-CAR. This retention of CAR in the CF can significantly decrease the solubility and reduce the loss of water vapor and TS, but increases the stiffness of the film. The films showed a tendency to yellowing, with a slight opacity and less WI than the CF. Furthermore, it was found that CF-CAR can block UV light. Furthermore, the CF-CAR presented antibacterial activity as measured by the agar diffusion method and antioxidant capacity. The CF showed a better ability to protect erythrocytes against oxidative attack. The improvement and conservation of bioactive components in the CF involve a step in the formation of new active packaging.